High-volume evacuation mitigates viral aerosol spread in dental procedures

Dental healthcare personnel (DHCP) are subjected to microbe-containing aerosols and splatters in their everyday work. Safer work conditions must be developed to ensure the functioning of the healthcare system. By simulating dental procedures, we aimed to compare the virus-containing aerosol generation of four common dental instruments, and high-volume evacuation (HVE) in their mitigation. Moreover, we combined the detection of infectious viruses with RT-qPCR to form a fuller view of virus-containing aerosol spread in dental procedures. The air–water syringe produced the highest number of aerosols. HVE greatly reduced aerosol concentrations during procedures. The air–water syringe spread infectious virus-containing aerosols throughout the room, while other instruments only did so to close proximity. Additionally, infectious viruses were detected on the face shields of DHCP. Virus genomes were detected throughout the room with all instruments, indicating that more resilient viruses might remain infectious and pose a health hazard. HVE reduced the spread of both infectious viruses and viral genomes, however, it did not fully prevent them. We recommend meticulous use of HVE, a well-fitting mask and face shields in dental procedures. We advise particular caution when operating with the air–water syringe. Due to limited repetitions, this study should be considered a proof-of-concept report.


Dental instruments
The procedures were performed using an air turbine handpiece (KaVo, Germany, Biberach), a high-speed dental handpiece (KaVo), an ultrasonic scaler G6-tip (NSK Varios 750), and an air-water syringe.The air-water syringe was used with air-water spray and with air only.The HVE applied was the dental unit's suction (Planmeca Compact i), with saliva evacuation at 80 ml/s.

Breathing simulation
A proportional valve-operated breathing simulation system was connected to the phantom head's nose via tygon tubing.The system provided approximately sinusoidal breathing cycles with 2.5 s of inflow (peak flow rate 24 lpm), 3 s of outflow (peak flow rate 16 lpm), and a break of 1 s.The tubing was attached to the nose, mimicking realistic flow jet directions and flow rates characterized by Gupta et al. [1].

Viruses, microbial strains, growth media and plates
Purified Phi6 was used as a model virus and Pseudomonas syringae pv.phaseolicola HB10Y (HB10Y) as its host.Phi6 was purified using the sucrose gradient method described by Bamford et al. [2].As Phi6 is human-safe, it enables the assessment of viral aerosol spread during dental procedures without any risk to the staff.Phi6 is a suitable mechanical model for SARS-CoV-2; it is an RNA virus of comparable size to SARS-CoV-2 (~ 80-100 nm), similarly enveloped by a lipid membrane, has spike proteins, and is also preferred in other similar studies, including studies of virus survival in human saliva microdroplets [3].Both Phi6 and P. syringae were originally obtained from Ann Vidaver, University of Nebraska [4].Agar plates containing HB10Y (HB10Y plate) were prepared by mixing 100 µl of bacteria grown overnight at room temperature (RT, 22°C) in Luria-Bertani-Lennox (LB) broth (Supplement Table 2) with 3 ml of LB soft agar and pouring it on LB agar plates.

Virus collection
HB10Y plates and empty petri dishes were placed throughout the room (Supplement Figure 2) to collect passively deposited aerosols.Plates were open only during the procedures."After" plates were opened immediately after the procedure for 15 min to monitor the number of viruses that were deposited from the air after the procedures.After sample collection, HB10Y plates were incubated overnight (22°C).The empty petri dishes were washed with 1 ml of HEPES (Supplement Table 3) and analyzed with RT-qPCR.Two 5 ml Biosamplers (SKC Inc.), two Button samplers (SKC Inc.), and a six-stage Andersen cascade impactor were used to actively collect aerosols.Biosampler 1 was filled with 5 ml of HEPES and Biosampler 2 with 5 ml of LB.A pump created an airflow of 12.5 l/min through the Biosamplers.The positions of the Biosamplers are shown in Supplement Figure 2.
The Andersen impactor was filled with HB10Y plates and was used to collect infectious viruses in aerosol droplets of different size ranges.A pump was adjusted to create a 28.3 l/min airflow through the collector.The airflows were calibrated using Mass Flowmeter 3063 (TSI Inc., USA, Minnesota City, MN).
The DHCP wore Button samplers on their chest during the procedures, which collected particles of <100 μm.The Button samplers were connected to Gilian 5000 airsampling pumps (Sensidyne, USA, St. Petersburg, FL) using a 4 l/min airflow.Mixed cellulose ester filters (1.2 µm, SKC Inc.) were used in the Button samplers.

Aerosol measurements
The total aerosol particle number concentration at diameters >10 nm was measured with a Condensation Particle Counter (CPC), model 3007 (TSI Inc.)The CPC flow rate was 0.8 l/min and a 1-s time resolution was used in data logging.The flow rate was validated using a calibrated Gillian flowmeter before and after the measurements and the data were corrected accordingly.
An Optical Particle Sizer (OPS) model 3330 (TSI Inc.), flow rate 1 l/min, was used to measure the aerosol particle number size distribution at the range 0.3-10 µm, divided into 16 channels.Flow rate was verified at laboratory settings prior and after the measurements, similar to the CPC.The OPS was operated at 10-s time resolution.The positions of the aerosol measurement devices are shown in Supplement Figure 2.

RT-qPCR
The RT-qPCR analysis was carried out according to Gregorova et al. [5].A sample volume of 5 μl was used.Data analysis was done using QuantStudio™ Design & Analysis Software v1.5.2.The method had a detection threshold of 2•10³ genome copies / ml, which was due to the small sample volume used in the protocol.